Zebrafish tsc1 and cxcl12a increase susceptibility to mycobacterial infection

Knockdown of miR-126 increases susceptibility to mycobacterial infection which can be independently reversed by targeting Tsc1/mTOR or ccr2 implicating macrophage function.

authors fail to extract RNA in sufficient quantity/quality?If so why?What was exactly tried (e.g.scaling up)?Which DEG were identified?This needs to be clarified, i.e. data need to be presented in the supplement.3. The reviewer asked for the original expression data in uninfected embryos (e.g.relative expression).These data are still missing.Therefore, the data are requested again as supplementary information together with an explanation on how the fold change for uninfected controls was calculated (e.g. in figure 2A-E).4. The reviewer asked for a further characterization of the macrophages (e.g. by iNOS).Any reply from the authors regarding this aspect is still missing.5.The quality of the graphical abstract (figure 11) is not suitable for this journal.Please provide a complete, visually appealling graphical abstract.
Additional minor aspects: Line 45 Please correct: mTOR inhibition.We find Figure 1 B The legend is missing.
Figure 1 E Please remove statistics where n = 2; this is not suitable to perform statistical analysis.Should not Scram inf show around a two-fold increase compared to Scram uninf (compare to 1A)? Line 373:wq Please correct: Our data thus far indicates

Report for Author:
This manuscript demonstrated that miR-126 upregulation protects the zebrafish embryonic mycobacterium model.Two possible miR-126 targets Tsc1 and Cxcl12, mediate mTOR signaling or macrophage recruitment and activation, respectively.Suppression of either one in miR-126 deficient condition resulted in reduced bacteria burden.Although the importance of mTOR and CCR2 signaling in Mm infection in zebrafish models has been previously reported, the direct connection of these two pathways to miR-126 and its possible target tsc1 and cxcl12 is novel.However, the current study's significant weaknesses still need to be addressed.Major concerns: 1.All experiments should be replicated three times.Power analysis should be used to determine the proper number of samples of zebrafish larvae.Instead of a range of larvae used in each experiment, the exact number should be specified.2. It would be essential to show the RNA sequencing data and related pathway analysis.Although the selected RNA level did not show any difference in the RNA sequencing experiment, it does not necessarily mean this result is invalid.Understanding the consequence of overall transcript changes upon miR-126 depletion is essential.3. The manuscript lacks a description or discussion of the expression pattern for miR-126 upon Mm infection.This information is essential to understand which cells, Tsc1 or cxcl12, exert their biological function.An alternative way is to overexpress miR-126 in specific tissue and determine its impact on the infection outcome.4, Figure 2 is problematic.tsc1a is the only one that showed significant upregulation upon miR-126 suppression.The remaining genes did not show significant changes upon Mm infection and miR-126 deletion.I suggest removing the non-significant data.Minor concerns: 1. Figure 1E.Why would miR-126 sgRNA injection reduce its mRNA level? 2. Figure 4 should include the transcript names and group them in functional groups in A and C to make the data useful.
3. Data of miR-126 target prediction should be better organized using, for example, a Venn diagram to show the overlap of the predicted and validated targets rather than displaying the raw search results.
Secondly, the claim that zebrafish TSC1a would likely be a miR-126 target because of homology and sequence analysis is not a solid foundation for a manuscript based on this.More detailed experiments need to be presented to confirm this.
If this cannot be provided, the data on TSC1 and miR-126 distinct roles in M. marinum host defence need to be discussed separately -possibly in 2 different papers

Referee #1 Review
Report for Author: Wright et al. show an interesting mechanistic link between miRNA-126 signalling and downstream targets in a M. marinum zebrafish model.The basic findings are that mycobacteria-induced miR-126 suppresses tsc1 and cxcl12a thereby improving macrophage function via mTOR signalling and the recruitment of TNF-expressing macrophages.The topic is interesting and of general relevance, and the potential of the study is very good.Yet, the work in its current form is incomplete.Thus, a thorough revision seems necessary to support the model as suggested.Major points: • Statistics: Data appear by in large not to be normally distributed, i.e. non-parametric tests and parameters are required.The statistical comparisons between conditions are incomplete and not properly depicted.All conditions of each experiment need to be compared and depicted similarly between panels, e.g. by tables below the figures, or by outlining in the legend, which comparisons reveals • It seems essential to see the original expression data in uninfected embryos, i.e. relative expression in uninfected controls related to house-keeping genes as data (e.g.Fig. 1A + B). • Fig. 1: It is stated "For qPCR analysis, each data point represents 10 embryos, and contains 2 biological replicates."The figure shows 4 data points.Does this mean that 2 of these data points are technical replicates and not biological replicates?If this is the case it is not possible to perform statistics.This aspect should be considered throughout the whole manuscript.Please clarify.Any technical replicates should be removed from the statistical analysis and at least one more biological replicate should be added.
• Figure 1d/2c/d etc.: How many embryos were analysed?Was there a power analysis?Why are numbers so variable (Fig. 2c)?In all legends: The exact number of analysed embryos has to be outlined for each condition (e.g.figure 7).
• Fig 2A : The bacterial load (cfu) for dESX as compared to wt M.marinum needs to be shown, the mutant is expected to be attenuated.• Fig. 3 and following: It is not sufficient (and up to date) to base transcriptional changes only on individual genes.RNAseq needs to be performed in embryos after miR-126 treatment (with and without infection) as well as Tsc1a ko embryos, and the alterations need to be depicted avoiding unnecessary bias.Thereby downstream genetic targets can be identified to allow for more stringency in interpreting in particular the "double knock out" conditions.
• While it is interesting to use rather robust markers for gene function like "bacterial area" and macrophages the precise intervention by CrispR gene ko and the read-out do not necessarily match in precision.In particular, the parameter "total bacterial area" needs to be backed up by conventional cfu counts.• Fig. 7 B: I am not convinced that "total neutrophil area at granuloma" is not different between scramble control and miR-126 ko late in infection, but total macrophage area at wound site is.This is complicated statistics.• Fig. 9 C. Since miR-126 in combination with tsc1a ko results in a mixed TNF response, the signalling axis is obviously more complicated than proposed.Again, this strongly argues for RNA sequencing analysis after miR-126 treatment and in tsc1a ko • Please provide a cartoon depicting your model of how miR-126, tsca interact in mycobacteria infection.

Additional points:
• Protective macrophages: This term should be avoided, if protection cannot be demonstrated by these macrophages specifically.It is unclear whether the macrophage area results from the bacterial count or vice versa.
• Classically activated macrophages may be a better term.Moreover, the cells need to be characterized more precisely, at least by staining for iNOS.
• Similarly: Permissive macrophages is a term that is not justified by the data.The wording on this matter should be much more cautious.
• mTOR signalling should be analysed with respect to the influence of rapamycin/MHY185 on macrophage recruitment.

Minor aspects
• Introduction (Lines 72 -78/Lines 89-90): In Lines 89/90 authors claim to analyse the mycobacterium-driven induction of miR-126.In Lines 72-78 they report examples of a reduction of miR-126 in different mycobacterial infections.For the reader this does not become clear at this point.Is miRNA-126 reduction in the examples described then rather a sign of uncontrolled or severe/disseminated forms of infection?It should be specified (for example comment if the infection dose of 200 CFU M. marinum in zebrafish is normally cleared or not, and therefore representative of a controlled or disseminated infection) or this aspect should be dealt with in the discussion alone (Lines 443 ff).In general, a comment on the course of M. marinum infection in zebrafish would be helpful for the reader to put the 1 day-and 3 day-infection in the right context.• Regarding the paper Swaim et al (doi: 10.1128/IAI.00887-06)200 CFU seem to be an intermediate infection dose.Please explain or discuss: Would a high-dose infection of zebrafish lead to a reduction of miRNA-126?Authors claim an important role of miRNA-126 in early time points of infection.Were later time points analyzed?Why were the timepoints and dosages chosen?• Method section: How did the table in Supplementary Figure 1 led to the selection of the finally analysed downstream targets?• Table headings are incomplete.
• Lines 153-154: Figure 3b does not show an increase of Cxcr4b in the knockdown embryo, there is a nonsignificant reduction.This needs to be corrected or explained.Or is Figure 3g referred to in this sentence?• Line 165/166: Is 3 dpi referred to (3J) or is the infected miR-126 knockdown meant here?In Figure 3e, there is no significant difference between miR-126 knockdown alone and infection alone.
• Figure 4c: The statistical analysis in this figure needs to be clarified (formatting problem).
• Line 216: Authors claim before that tsc1a is a negative regulator of mTOR, so knockdown of miR126 would decrease mTOR.Why is there now the opposite described in line 216?• Line 234: As noticed before the increase in Cxcr4b is only visible at day 3 pi (figure 3).This should be elaborated on.
• Figure 7A and 7C: Apparently, in 7A only the situation in infection is analysed.Yet, according to the figure legend, in 7C uninfected animals were also analysed.Where are those depicted?As authors labelled all conditions with dpi (days post infection) in 7C, the labelling should be controlled.Alternatively, the abbreviation dpi could be used as 'days post injection' (line 98) instead.Why are uninfected controls not necessary in 7A but in 7C?
• Line 354: Please correct: Knockdown of down both cxcl12a and ccr2 was protective, decreasing the number of TUNEL-stained cells at 3 dpi.In general, all of the data and not representative plots should be depicted (except for imaging of course).Also, for example in Figure 8 the whole data set needs to be depicted.

Referee #2 Review
Report for Author: This is an exciting story characterizing the biological function of miR-126 in the zebrafish Mm model.The authors showed convicting evidence that miR-126 is protective, specifically in virulent Mm infection.Suppressing miR-126 at the whole organism level resulted in increased cell death, increased macrophage recruitment to the bacteria, reduced macrophage activation, and increased bacterial burden.Through miR-126 target discovery, they found tsc-1 and cxcl12a as the two functional relevant miR-126 targets.Tsc-1 and related mTOR pathways regulate cell death and macrophage activation.At the same time, cxcl12a promotes ccl2 expression and activates ccr2 to regulate macrophage recruitment, activation, and cell death.At the same time, the role of mTOR in the zebrafish Mm infection model is known.The link with miR-126 is novel.Regarding ccr2, it is not playing a significant role in this model unless miR-126 is depleted.This result is interesting, given that human patients have reduced miR-126 serum levels.However, there are several issues related to the inclusion of the controls or the interpretation of the data which should be addressed before the manuscript can be accepted for publication.Major concerns: 1.In several places, such as figure 1, the authors used uninjected control for the miR-126 knockdown.Since the knockdown uses antagomir, which is essentially nucleotide. it is important to include the scramble control to rule out possible activation due to nucleotide injection.2. Whereas using target prediction software or the database of known miR-126 targets can give some clue, a better approach is to use RNA seq to identify miR-126 regulated gene transcription network under the infection condition, which will allow an unbiased comprehensive understanding of miR-126 regulated pathways.3. The evidence that tsc1 and cxcl12a are the direct miR-126 targets is missing.4. In figure 5, although increased mTOR activation is evident, however it is not clear in which cells the mTOR is activated.It would be informative to determine whether the pS6 positive cells are macrophages.5. Same with Figure 6, are the cells with increased TUNEL staining macrophages?6.From the data presented in Figure 10, looks like all phenotype resulting from miR-126 knockdown can be rescued with ccr2 knockdown.Will knocking down ccr2 and tsc1 together in this situation have addictive effects? 7. line 264, the authors concluded that miR-126 does not influence macrophage production.However, the macrophage number in CHT is reduced, which is quite the opposite of the conclusion.8. Line 312, as mentioned above, the current manuscript lacks evidence that cell death happens in macrophages.9. line 417-421.The author implied that miR-126 depletion increase ccr2+ macrophage numbers.However, the data presented in this manuscript investigated the ccr2 levels in the entire organism.Is ccr2 only expressed in macrophages?10. line 432-442, the authors discussed the possible involvement of spred1 as a miR-126 target.however, the data in Figure 3 D, I did not show a significant regulation of spred1 by miR-126 in infection.Therefore, spred1 is not relevant to miR-126 in the current model and this paragraph should be removed or rewritten.
Minor concerns: 1.It would be interesting to figure out the expression pattern of miR-126 during Mm infection.Is miR-126 expressed in the hematopoietic or non-hematopoietic tissues? 2. I would suggest organizing the paper a bit differently.Figure 9CD should be moved to Figure 8-both looking at tsc1 and macrophages.

Referee #3 Review
Report for Author: This study by Wright et al describes a protective role for microRNA miR-126 in zebrafish infection with the pathogenic mycobacterium, M. marinarum, which models human tuberculosis.To explain this observation mechanistically, the authors screen for potential mRNA targets of miR-126 involved in the host response to infection.They uncover some interesting targets including a negative regulator of mTOR, Tscl1.Although some of the bacteria-permissive effects of miR-126 ablation are reverse in Tscl1 "knockdown" animals and ablated by Tscl1 disruption, when examining the role of mTOR in this context, mTOR and Tscl1 independent roles are revealed, linked to miR-126.Notably, these include an effect on chemokine signalling, which triggers the recruitment of mycobacteria-permissive macrophages in the absence of miR-126 and facilitates infection.This data is interesting as it reveals a key host defence mechanism to limit this bacteria-favoring response during infection, which could be relevant to human TB, but because of the compounding effects of miR-126 knockdown alongside target gene ablation, it is still unclear which is the central molecular target for miR-126 mediating its protective effects.Further molecular work defining this target would be required to address this & may simplify the key finding and make this study more relevant to the wider molecular biology audience, as at present its relevance and interest may be quite limited to the host/pathogen field.Some major points are outlined below; TSCL1 ablation model; The authors describe the targeting of Tscl1; "we targeted tsc1a for knockdown using CRISPR-Cas9 (Figure 4B).Knockdown of tsc1a significantly reduced transcript abundance".Although some sequence information is provided in Table 1, not much extra information is provided to assess what is occurring in this animal, which is crucial to interpretation of much of the subsequent work and observed phenotypes.Is this a knock-out, ie is the coding sequence for Tscl1 removed?Or is there less RNA made/translated into protein in this model?Judging from data in Fig4C, while Tscl1 RNA is still present, there is less in the "Tscl1 knockdown", which is reduced further with M.marinarum infection.This reviewer wonders if some of the inconsistencies among the different phenotypes measured (pS6-activity, bacterial burden, cell death, macrophage recruitment, TNF promoter activation, CXCL12 induction etc) is because of a tonic level of Tscl1 still present in the "knock-down" which may regulate some of these phenotypes to the same extent as wild-type, but not all?Crucially, although the authors nicely demonstrate mTOR-independent effects of Tscl1 linked to cell death and bacterial dissemination, TSCL1 does not regulate CCR2+ macrophage recruitment -the cells which allow bacterial replication in this model.Would this phenotype be different in a TSCL1-deficient model?A specific zebrafish could be generated where miR-126 targeting of the Tscl1 3'UTR can directly be targeted -using morpholino target protectors or specific editing of the 3'UTR coding sequence?At a minimum, further information on the levels and activity of TSCL1 in the "knockdown" model should be provided to allow better interpretation of this data.However, a clearer way to ablate/disrupt TSCL1 could allow better clarity among some of the observed effects.
Cellular Heterogeneity; Although Tscl1 targeting reduces some of the effects of miR-126 inhibition, like bacterial survival and cell death, it does not inhibit macrophage recruitment of CCR2+ cells.Is this cell death observed in the Tscl1 knockdown animal occurring in a different cellular population than the CCR2+ recruited cells, which are recruited in response to the cell death?And is it likely then that miR-126 controls expression of different targets in both macrophage/cellular populations?TSCL1 in the "resident" population and a separate target linked to chemokine signalling in the CCR2+ cells?Direct miR-126 Targets; This links to my final major point.The authors use bioinformatic analysis to predict miR-126 targets involved in the host response to mycobacterial infection in the zebrafish model.An initial mRNA screen validates some of these.Tscl1 emerges as a strong candidate and an RNA-Hybrid plot is provided to illustrate the potential interaction.3'UTR Luciferase Assays should be employed to confirm this, in a reporter cell system.Additionally, it is unclear from the manuscript if CXCL12a or CCL2/CCR2 are thought to be direct targets of miR126 or their expression is regulated by other targets (the Tscl1/mTOR axis?).3'UTR Luciferase assays would strengthen this and may point to the most relevant targets for miR-126.
Minor points for correction/clarification; Intro; line 58/59; would it be better to describe microRNAs as "fine-tuners" rather than "master regulators" Line 72/73; any information on which cells in cattle decreased miR-126 was observed in Line 75/76; similarly, what cells or tissues in humans?Results,Fig 7c: in the recruited macrophage reporter mouse, the trace does not appear to show increased recruitment in control (non-knockdown) mice, is this expected?Remarks To Editors; This is a well performed and interesting study in a model of infection relevant to human disease, but which can be more easily targeted to reveal novel mechanistic insight.In this case, much has been revealed about miR-126 biology, however, some of this work remains incomplete -particularly the observed Tscl1-independent effects on macrophage recruitment, which seem to be central to the protective effects of miR-126/bacteriapermissive effects of miR-126 inhibition.the distinctions between these 2 pathways need to be more clearly delineated and the following points should be addressed in any potential revision for this journal.
-Clarification on the role of TSCL1 and methods to ablate -More specific ablation or targeting of the miR-126/TSCL1 interaction would be desirable -3'UTR Target Analysis to define direct miR-126 molecular targets -Is there differences in miR126 targeting in different macrophage/ceullular populations?
Authors' Response to Reviewers Referee #1: Wright et al. show an interesting mechanistic link between miRNA-126 signalling and downstream targets in a M. marinum zebrafish model.The basic findings are that mycobacteria-induced miR-126 suppresses tsc1 and cxcl12a thereby improving macrophage function via mTOR signalling and the recruitment of TNFexpressing macrophages.The topic is interesting and of general relevance, and the potential of the study is very good.Yet, the work in its current form is incomplete.Thus, a thorough revision seems necessary to support the model as suggested.
Major points: • Statistics: Data appear by in large not to be normally distributed, i.e. non-parametric tests and parameters are required.The statistical comparisons between conditions are incomplete and not properly depicted.All conditions of each experiment need to be compared and depicted similarly between panels, e.g. by tables below the figures, or by outlining in the legend, which comparisons reveals Non-normally disturbed data have been re-analysed using non-parametric tests and all statistical comparisons displayed on each graph.The methods sub-heading "statistics" has been updated to include precise tests used.
• It seems essential to see the original expression data in uninfected embryos, i.e. relative expression in uninfected controls related to house-keeping genes as data (e.g.Fig. 1A + B).
• Fig. 1: It is stated "For qPCR analysis, each data point represents 10 embryos, and contains 2 biological replicates."The figure shows 4 data points.Does this mean that 2 of these data points are technical replicates and not biological replicates?If this is the case it is not possible to perform statistics.This aspect should be considered throughout the whole manuscript.Please clarify.Any technical replicates should be removed from the statistical analysis and at least one more biological replicate should be added.
Biological replicates are now plotted across all qPCR analyses.
• Figure 1d/2c/d etc.: How many embryos were analysed?Was there a power analysis?Why are numbers so variable (Fig. 2c)?In all legends: The exact number of analysed embryos has to be outlined for each condition (e.g.figure 7).Number of embryos is now stated in legends.No power analysis was performed.
To be frankly honest if we could make the infections less variable, we would.We assume the zebrafish embryo yields more variable burden data than cell culture and mouse experiments because they are genetically diverse (not inbred) and there is additional variation introduced by the micromanipulation procedure to inject bacteria.
• Fig 2A : The bacterial load (cfu) for dESX as compared to wt M.marinum needs to be shown, the mutant is expected to be attenuated.
Yes, the mutant is attenuated.Since we do not have matched infection burdens we have removed Figure 2 entirely.
• Fig. 3 and following: It is not sufficient (and up to date) to base transcriptional changes only on individual genes.RNAseq needs to be performed in embryos after miR-126 treatment (with and without infection) as well as Tsc1a ko embryos, and the alterations need to be depicted avoiding unnecessary bias.Thereby downstream genetic targets can be identified to allow for more stringency in interpreting in particular the "double knock out" conditions.RNAseq analysis of Tsc1a KD showed an increase in ribosomal biogenesis consistent with relieving suppression of TOR and an increase in immune gene expression consistent with our phenotype of reduced bacterial burden.This data is presented in Figure 4 and described in the results section around Line 200.
We made two attempts at RNAseq analysis of miR-126 knockdown embryos which generated only very truncated lists of DEGs.Surprisingly despite using RNA samples (generated from two countries) that showed differential expression of tsc1a, cxcr4b, and cxcl12a by our conventional cDNA synthesis/qPCR measurement, these transcripts were all found to be present at equivalent levels by RNAseq measurement.In light of this discrepancy we have rewritten the initial sections to include mention of the uncertainty of a miR-126/tsc1a axis and to refocus the writing of the overall manuscript to focus on the downstream tsc1a and cxcl12a phenotypes.See lines 172 and 500 for specific mention of this failure.
• While it is interesting to use rather robust markers for gene function like "bacterial area" and macrophages the precise intervention by CrispR gene ko and the read-out do not necessarily match in precision.In particular, the parameter "total bacterial area" needs to be backed up by conventional cfu counts.
New data is presented in Supplementary Figure 1 showing a replication of the critical experiment from Figure 1D with measurements by fluorescent pixel count and CFU recovery demonstrating the same pattern of bacterial burden across both measurement techniques.We also direct the reviewer to Walton et al, Cell Host Microbe 2018 (PMID: 30308157) for data showing strong correlation of fluorescent pixel count burden estimation with RT-qPCR measurement of bacterial 16S rRNA as a separate recent validation of fluorescent pixel count compared to a third measurement technique.
• Fig. 7 B: I am not convinced that "total neutrophil area at granuloma" is not different between scramble control and miR-126 ko late in infection, but total macrophage area at wound site is.This is complicated statistics.
Although there may in fact be a measurable difference at that particular timepoint, this difference is not statistically significant by the, admittedly simple, Sidak correction ANOVA test that we utilised.We are open to the reviewer's suggestion for a more appropriate test to perform multiple comparisons across treatment groups and along a timeseries.
• Fig. 9 C. Since miR-126 in combination with tsc1a ko results in a mixed TNF response, the signalling axis is obviously more complicated than proposed.Again, this strongly argues for RNA sequencing analysis after miR-126 treatment and in tsc1a ko This project has been unfunded since the end of 2021 and additional RNAseq validation of qPCR results is not possible.
• Please provide a cartoon depicting your model of how miR-126, tsca interact in mycobacteria infection.
We have added a cartoon as Figure 11.
Additional points: • Protective macrophages: This term should be avoided, if protection cannot be demonstrated by these macrophages specifically.It is unclear whether the macrophage area results from the bacterial count or vice versa.
• Classically activated macrophages may be a better term.Moreover, the cells need to be characterized more precisely, at least by staining for iNOS.
• Similarly: Permissive macrophages is a term that is not justified by the data.The wording on this matter should be much more cautious.
Wording has been revised to "tnfa expressing" to reflect the technical assay used in this study.
• mTOR signalling should be analysed with respect to the influence of rapamycin/MHY185 on macrophage recruitment.
The macrophage migration assay was repeated with rapamycin/MHY1485 treatment demonstrating the increased migration phenotype is mTOR-independent (Figure 8).

Minor aspects:
• Introduction (Lines 72 -78/Lines 89-90): In Lines 89/90 authors claim to analyse the mycobacteriumdriven induction of miR-126.In Lines 72-78 they report examples of a reduction of miR-126 in different mycobacterial infections.For the reader this does not become clear at this point.Is miRNA-126 reduction in the examples described then rather a sign of uncontrolled or severe/disseminated forms of infection?It should be specified (for example comment if the infection dose of 200 CFU M. marinum in zebrafish is normally cleared or not, and therefore representative of a controlled or disseminated infection) or this aspect should be dealt with in the discussion alone (Lines 443 ff).In general, a comment on the course of M. marinum infection in zebrafish would be helpful for the reader to put the 1 day-and 3 day-infection in the right context.
Added a description of our model as "acute disseminated mycobacterial infection" in introduction (line 127) and discrepancy between experimental infection increase in zebrafish miR-126 vs reduced human miR-126 in tuberculosis in discussion (line 623).
• Regarding the paper Swaim et al (doi: 10.1128/IAI.00887-06)200 CFU seem to be an intermediate infection dose.Please explain or discuss: Would a high-dose infection of zebrafish lead to a reduction of miRNA-126?Authors claim an important role of miRNA-126 in early time points of infection.Were later time points analyzed?Why were the timepoints and dosages chosen?200 CFU is used as a standard dose for zebrafish embryos (doi: 10.1093/infdis/jiw355 & doi: 10.1038/nature13967) which results in a progressive granulomatous infection.The infection experiments performed by Swaim et.al are performed on adult zebrafish and injected intraperitoneally.Our infection model utilises lower doses due to the size and age of the embryos, as well as the injection of bacteria directly into the circulation or musculature.
Later timepoints could not be analysed for our original experimental set up as the antagomir diluted out after 4-5 days post fertilisation / 3-4 days post infection.
• Method section: How did the table in Supplementary Figure 1 led to the selection of the finally analysed downstream targets?
The methods subsection "miRNA target prediction" has been updated to include selection criteria for downstream targets.
• Table headings are incomplete.
• Lines 153-154: Figure 3b does not show an increase of Cxcr4b in the knockdown embryo, there is a nonsignificant reduction.This needs to be corrected or explained.Or is Figure 3g referred to in this sentence?
Wording has been updated to clarify this… "From the gene expression analysis, cxcr4b, cxcl12a, and tsc1a were regarded as potential target genes of miR-126 due to their increased expression in knockdown and knockdown infected embryos and differential regulation compared to M. marinum infected embryos.Despite no increase in expression at 1 dpi of cxcr4b in miR-126 knockdown alone, the increased expression in miR-126 knockdown infected embryos made cxcr4b a gene of interest alongside cxcr4a and cxcl12a due to their previously identified role in mycobacterial and zebrafish immunity…" • Line 165/166: Is 3 dpi referred to (3J) or is the infected miR-126 knockdown meant here?In Figure 3e, there is no significant difference between miR-126 knockdown alone and infection alone.
Text has been corrected and updated to clarify significant differences.
• Figure 4c: The statistical analysis in this figure needs to be clarified (formatting problem).
Formatting of statistical comparisons has been corrected.
• Line 216: Authors claim before that tsc1a is a negative regulator of mTOR, so knockdown of miR126 would decrease mTOR.Why is there now the opposite described in line 216?
Incorrect wording (increased mTOR activity) has been corrected to "decreased mTOR activity".
• Line 234: As noticed before the increase in Cxcr4b is only visible at day 3 pi (figure 3).This should be elaborated on.
• Figure 7A and 7C: Apparently, in 7A only the situation in infection is analysed.Yet, according to the figure legend, in 7C uninfected animals were also analysed.Where are those depicted?As authors labelled all conditions with dpi (days post infection) in 7C, the labelling should be controlled.Alternatively, the abbreviation dpi could be used as 'days post injection' (line 98) instead.Why are uninfected controls not necessary in 7A but in 7C?
Labelling has been updated to include designation of uninfected and infected embryos and improve readability of graphs.Uninfected controls have been included for Figure 7A.
• Line 354: Please correct: Knockdown of down both cxcl12a and ccr2 was protective, decreasing the number of TUNEL-stained cells at 3 dpi.In general, all of the data and not representative plots should be depicted (except for imaging of course).Also, for example in Figure 8 the whole data set needs to be depicted.
Wording has been corrected from biological replicates to experimental replicates.
---------------Referee #2: This is an exciting story characterizing the biological function of miR-126 in the zebrafish Mm model.The authors showed convicting evidence that miR-126 is protective, specifically in virulent Mm infection.Suppressing miR-126 at the whole organism level resulted in increased cell death, increased macrophage recruitment to the bacteria, reduced macrophage activation, and increased bacterial burden.Through miR-126 target discovery, they found tsc-1 and cxcl12a as the two functional relevant miR-126 targets.Tsc-1 and related mTOR pathways regulate cell death and macrophage activation.At the same time, cxcl12a promotes ccl2 expression and activates ccr2 to regulate macrophage recruitment, activation, and cell death.At the same time, the role of mTOR in the zebrafish Mm infection model is known.The link with miR-126 is novel.Regarding ccr2, it is not playing a significant role in this model unless miR-126 is depleted.This result is interesting, given that human patients have reduced miR-126 serum levels.However, there are several issues related to the inclusion of the controls or the interpretation of the data which should be addressed before the manuscript can be accepted for publication.
Major concerns: 1.In several places, such as figure 1, the authors used uninjected control for the miR-126 knockdown.Since the knockdown uses antagomir, which is essentially nucleotide. it is important to include the scramble control to rule out possible activation due to nucleotide injection.
All control embryos used for miR-126 antagomiR knockdown and Crispr knockdown experiments were injected with a corresponding scramble control sequence.Additional experimental data using a scramble gRNA/Cas9 vs miR-126 gRNA/Cas9 injected F0 crispant system have been added in Figure 1E and F demonstrating a second line of evidence that miR-126 is hostprotective against M. marinum infection.
2. Whereas using target prediction software or the database of known miR-126 targets can give some clue, a better approach is to use RNA seq to identify miR-126 regulated gene transcription network under the infection condition, which will allow an unbiased comprehensive understanding of miR-126 regulated pathways.
3. The evidence that tsc1 and cxcl12a are the direct miR-126 targets is missing.
Both Tsc1 and Cxcl12a have previously been identified as direct targets of miR-126.A study in zebrafish has shown direct targeting of Cxcl12a by miR-126a (doi: 10.1161/ATVBAHA.116.308120) while multiple studies have identified Tsc1 as a target of miR-126 in mice, humans, and pigs (doi: 10.1038/ni.2767)(doi: 10.1007/s11626-018-0292-0).As the degree of sequence homology and apparent conservation of target genes is high, we believe that Tsc1 is a direct target of miR-126 in zebrafish.This has been further mentioned in the text.
We made two attempts at RNAseq analysis of miR-126 knockdown embryos which generated only very truncated lists of DEGs.Surprisingly despite using RNA samples (generated from two countries) that showed differential expression of tsc1a, cxcr4b, and cxcl12a by our conventional cDNA synthesis/qPCR measurement, these transcripts were all found to be present at equivalent levels by RNAseq measurement.Furthermore, we were unable to perform experiments showing direct binding of zebrafish miR-126 to anything in cell culture transfection experiments using the predicted target sequence as bait in dual luciferase assays.
In light of these failures we have rewritten the initial sections to include mention of the uncertainty of a miR-126/tsc1a axis and to refocus the writing of the overall manuscript to focus on the downstream tsc1a and cxcl12a phenotypes.4. In figure 5, although increased mTOR activation is evident, however it is not clear in which cells the mTOR is activated.It would be informative to determine whether the pS6 positive cells are macrophages.
We have assessed the distribution and localisation of pS6 staining in macrophage reporter lines showing minimal colocalisation or specificity to macrophages.5. Same with Figure 6, are the cells with increased TUNEL staining macrophages?
We have included images of TUNEL staining in macrophage reporter lines showing colocalisation of TUNEL positive cells with bacteria and macrophages.
6. From the data presented in Figure 10, looks like all phenotype resulting from miR-126 knockdown can be rescued with ccr2 knockdown.Will knocking down ccr2 and tsc1 together in this situation have addictive effects?
This is an interesting idea to look at interaction between the pathways.We attempted to perform combinations of triple knockdown of miR-126/ccr2/tsc1a.Unfortunately, our embryos did not develop normally which we think is due to too much injected material (antagomir and gRNA/Cas9 complexes).Our live imaging data in Figure 8 provides evidence of independence as manipulation of tsc1/mTOR did not affect macrophage migration phenotypes in the control and miR-126 knockdown backgrounds.7. line 264, the authors concluded that miR-126 does not influence macrophage production.However, the macrophage number in CHT is reduced, which is quite the opposite of the conclusion.
Our data shows reduced macrophage numbers in the CHT only in the context of infection where there is increased recruitment from the CHT to the sites of infection.Thus we hypothesise that rather than decreased production of macrophages, and that the production of new cells is not able to "keep up" with the recruitment requirements (Pagan 2015 Cell Host and Microbe PMID: 26159717).
A detailed study of the role of miR-126 in myelopoiesis/monocyte differentiation will be grounds for future study.We have updated the text to clarify that there is in fact a difference in the numbers and explain our theory.
8. Line 312, as mentioned above, the current manuscript lacks evidence that cell death happens in macrophages.
As above, we have included images with macrophages, bacteria, and TUNEL staining.9. line 417-421.The author implied that miR-126 depletion increase ccr2+ macrophage numbers.However, the data presented in this manuscript investigated the ccr2 levels in the entire organism.Is ccr2 only expressed in macrophages?
According to a publicly available single cell RNAseq dataset of dissected granulomas from Cronan et al Cell 2021, ccr2 is expressed broadly by macrophages and dendritic cells.Expression was absent in neutrophils, B and T cells, and RBCs.
We have attempted to perform whole mount in situ hybridisation with our existing reagents to confirm these datasets but were unable to observe clean staining with our probes.10. line 432-442, the authors discussed the possible involvement of spred1 as a miR-126 target.however, the data in Figure 3 D, I did not show a significant regulation of spred1 by miR-126 in infection.Therefore, spred1 is not relevant to miR-126 in the current model and this paragraph should be removed or rewritten.Discussion relating to Spred1 has been removed.
Minor concerns: 1.It would be interesting to figure out the expression pattern of miR-126 during Mm infection.Is miR-126 expressed in the hematopoietic or non-hematopoietic tissues?miR-126 appears to be expressed fairly ubiquitously.For instance we saw mTOR activity effects in stromal cells in addition to the effects on haematopoietic cell phenotypes which we have focused on and the blood vessel phenotypes documented in the literature.
2. I would suggest organizing the paper a bit differently.Figure 9CD should be moved to Figure 8-both looking at tsc1 and macrophages.
The additional mTOR modulation data in Figure 8 makes the new Figure 8 quite large already.
---------------Referee #3: This study by Wright et al describes a protective role for microRNA miR-126 in zebrafish infection with the pathogenic mycobacterium, M. marinarum, which models human tuberculosis.To explain this observation mechanistically, the authors screen for potential mRNA targets of miR-126 involved in the host response to infection.They uncover some interesting targets including a negative regulator of mTOR, Tscl1.Although some of the bacteria-permissive effects of miR-126 ablation are reverse in Tscl1 "knockdown" animals and ablated by Tscl1 disruption, when examining the role of mTOR in this context, mTOR and Tscl1 independent roles are revealed, linked to miR-126.Notably, these include an effect on chemokine signalling, which triggers the recruitment of mycobacteria-permissive macrophages in the absence of miR-126 and facilitates infection.This data is interesting as it reveals a key host defence mechanism to limit this bacteriafavoring response during infection, which could be relevant to human TB, but because of the compounding effects of miR-126 knockdown alongside target gene ablation, it is still unclear which is the central molecular target for miR-126 mediating its protective effects.Further molecular work defining this target would be required to address this & may simplify the key finding and make this study more relevant to the wider molecular biology audience, as at present its relevance and interest may be quite limited to the host/pathogen field.Some major points are outlined below; TSCL1 ablation model: The authors describe the targeting of Tscl1; "we targeted tsc1a for knockdown using CRISPR-Cas9 (Figure 4B).Knockdown of tsc1a significantly reduced transcript abundance".Although some sequence information is provided in Table 1, not much extra information is provided to assess what is occurring in this animal, which is crucial to interpretation of much of the subsequent work and observed phenotypes.Is this a knockout, ie is the coding sequence for Tscl1 removed?Or is there less RNA made/translated into protein in this model?Judging from data in Fig4C, while Tscl1 RNA is still present, there is less in the "Tscl1 knockdown", which is reduced further with M.marinarum infection.This reviewer wonders if some of the inconsistencies among the different phenotypes measured (pS6-activity, bacterial burden, cell death, macrophage recruitment, TNF promoter activation, CXCL12 induction etc) is because of a tonic level of Tscl1 still present in the "knock-down" which may regulate some of these phenotypes to the same extent as wild-type, but not all?Crucially, although the authors nicely demonstrate mTOR-independent effects of Tscl1 linked to cell death and bacterial dissemination, TSCL1 does not regulate CCR2+ macrophage recruitment -the cells which allow bacterial replication in this model.Would this phenotype be different in a TSCL1-deficient model?A specific zebrafish could be generated where miR-126 targeting of the Tscl1 3'UTR can directly be targeted -using morpholino target protectors or specific editing of the 3'UTR coding sequence?At a minimum, further information on the levels and activity of TSCL1 in the "knockdown" model should be provided to allow better interpretation of this data.However, a clearer way to ablate/disrupt TSCL1 could allow better clarity among some of the observed effects.
The use of a tsc1a null mutant would be the ideal response to this concern.Our additional data in Figure 4 confirms a strong effect of tsc1a knockdown so while there may be residual tonic levels of Tsc1a active throughout the embryo we.Figure 11 summarises our findings which point toward distinct tsc1a and ccr2 mediated mechanisms of susceptibility that have overlapping measurable phenotypes (bacterial burden, cell death, tnfa-expressing cells).
Cellular Heterogeneity: Although Tscl1 targeting reduces some of the effects of miR-126 inhibition, like bacterial survival and cell death, it does not inhibit macrophage recruitment of CCR2+ cells.Is this cell death observed in the Tscl1 knockdown animal occurring in a different cellular population than the CCR2+ recruited cells, which are recruited in response to the cell death?And is it likely then that miR-126 controls expression of different targets in both macrophage/cellular populations?TSCL1 in the "resident" population and a separate target linked to chemokine signalling in the CCR2+ cells?This is a very interesting idea, however the zebrafish is not well suited to answer this particular question as there are (to our knowledge) no tools to track CCR2+ zebrafish cells.The best available technologies (RNAscope) are post mortem techniques that do not allow dynamic tracking and differentiation of responsive/resident cell populations.Our new data in Figure 8 showing independence of tsc1a/mTOR activity (measured as protection from cell death) from the ccr2-mediated migration phenotype (and any macrophage migration phenotype in the control animals) suggest the simple zebrafish embryo macrophage population may be fairly homogenous in this regard.
Direct miR-126 Targets: This links to my final major point.The authors use bioinformatic analysis to predict miR-126 targets involved in the host response to mycobacterial infection in the zebrafish model.An initial mRNA screen validates some of these.Tscl1 emerges as a strong candidate and an RNA-Hybrid plot is provided to illustrate the potential interaction.3'UTR Luciferase Assays should be employed to confirm this, in a reporter cell system.Additionally, it is unclear from the manuscript if CXCL12a or CCL2/CCR2 are thought to be direct targets of miR126 or their expression is regulated by other targets (the Tscl1/mTOR axis?).3'UTR Luciferase assays would strengthen this and may point to the most relevant targets for miR-126.
Both Tsc1 and Cxcl12a have previously been identified as direct targets of miR-126.A study in zebrafish has shown direct targeting of Cxcl12a by miR-126a (doi: 10.1161/ATVBAHA.116.308120) while multiple studies have identified Tsc1 as a target of miR-126 in mice, humans, and pigs (doi: 10.1038/ni.2767)(doi: 10.1007/s11626-018-0292-0).As the degree of sequence homology and apparent conservation of target genes is high, we believe that Tsc1 is a direct target of miR-126 in zebrafish.This has been further mentioned in the text.
We made two attempts at RNAseq analysis of miR-126 knockdown embryos which generated only very truncated lists of DEGs.Surprisingly despite using RNA samples (generated from two countries) that showed differential expression of tsc1a, cxcr4b, and cxcl12a by our conventional cDNA synthesis/qPCR measurement, these transcripts were all found to be present at equivalent levels by RNAseq measurement.Furthermore, we were unable to perform experiments showing direct binding of zebrafish miR-126 to anything in cell culture transfection experiments using the predicted target sequence as bait in dual luciferase assays.
In light of these failures we have rewritten the initial sections to include mention of the uncertainty of a miR-126/tsc1a axis and to refocus the writing of the overall manuscript to focus on the downstream tsc1a and cxcl12a phenotypes.
Minor points for correction/clarification: Intro; line 58/59; would it be better to describe microRNAs as "fine-tuners" rather than "master regulators" Changed to "fine-tuners" Line 72/73; any information on which cells in cattle decreased miR-126 was observed in Line 75/76; similarly, what cells or tissues in humans?miR-126 was decreased in serum from cattle, while human and mouse studies used either serum/plasma/CSF or PBMC/THP-1 macrophages to assess miRNA expression.miR-126 was believed to be specifically expressed in endothelial cells, however it is likely that while much of the miR-126 is from these endothelial cells, during infection or exposure to pathogens, it is also produced by immune cells such as dendritic cells and macrophages.
Results, Fig 7c : in the recruited macrophage reporter mouse, the trace does not appear to show increased recruitment in control (non-knockdown) mice, is this expected?Yes, our data shows the timepoints we looked at have a plateau of macrophage recruitment to nascent granulomas in the control animals.This is quite interesting to think about in hindsight as we genuinely did not notice it.A quick literature review shows this is to be expected with relatively little change in macrophage migration over a 2 hour period reported by Hu et al Frontiers in Immunology 2023, and a 4 hour period shown in Saelens, Sweeney, and Viswanathan et al Cell 2022.Our videos demonstrate the process is as dynamic as expected for later growing granulomas but the overall number of macrophages appears to be balanced during this earliest stage of granuloma formation.To upload the revised version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plexYou will be guided to complete the submission of your revised manuscript and to fill in all necessary information.Please get in touch in case you do not know or remember your login name.
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--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-sciencealliance.org/copyright-license-feeB. MANUSCRIPT ORGANIZATION AND FORMATTING: Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- ------------------------------------------------------------------------- The authors have address most, however not all of the concerns, some not even with arguments (which is at least surprising given the long revision time).We appreciate the attempts to improve the paper with RNA sequencing.Due to rather mysterious methodological difficulties, no results could be obtained for sequencing of miR-126 knockdown embryos and the manuscript was reoriented with a focus on the impact on tsc1 and cxcl12 instead.Several major issues remain, some of which were already pointed out in the first round of reviews.
1.Although the authors failed to prove the existence of a direct miR-126/tsc1 axis and realigned the manuscript accordingly, this change in overall direction is not reflected by the current title.This needs to be changes Title has been changed to "Zebrafish tsc1 and cxcl12a increase susceptibility to mycobacterial infection".
2. The apparently uninterpretable RNA seq analysis remains a mystery ("very truncated lists of DEGs").Did the authors fail to extract RNA in sufficient quantity/quality?If so why?What was exactly tried (e.g.scaling up)?Which DEG were identified?This needs to be clarified, i.e. data need to be presented in the supplement.We gone back to our initial RNAseq dataset from 2021 to create Supplementary Figure 2 to cover this dataset because if gives the most DEGs.Raw sequencing information has been deposited in GEO.Subsequent RNAseq replicates of the same conditions yielded fewer DEGs and aggregation alongside this initial run further depleted the list of DEGs.We attribute this diminishing returns to expiry of knockdown and molecular biology reagents after the primary lab group closed.
We note that our initial RNAseq dataset confirm the known roles for miR-126 in controlling neurobiology and Notch signalling (haematopoiesis and blood vessel formation).
3. The reviewer asked for the original expression data in uninfected embryos (e.g.relative expression).These data are still missing.Therefore, the data are requested again as supplementary information together with an explanation on how the fold change for uninfected controls was calculated (e.g. in figure 2A-E).Original request: "It seems essential to see the original expression data in uninfected embryos, i.e. relative expression in uninfected controls related to house-keeping genes as data (e.g.Fig. 1A + B)." New text added to materials and methods, we think this clarifies the relative quantification calculation method and conversion to fold change: U6 or β-actin was used as an endogenous control for normalisation and data analysed using the 2 -ΔΔ Ct method with an average of controls used to set baseline and fold change was calculated as the ratio of the difference divided by the original value.
4. The reviewer asked for a further characterization of the macrophages (e.g. by iNOS).Any reply from the authors regarding this aspect is still missing.5.The quality of the graphical abstract (figure 11) is not suitable for this journal.Please provide a complete, visually appealling graphical abstract.
Ouch, this one felt personal.Graphical abstracts are optional at LSA so it has been removed.
Additional minor aspects: Line 45 Please correct: mTOR inhibition.We find Corrected This manuscript demonstrated that miR-126 upregulation protects the zebrafish embryonic mycobacterium model.Two possible miR-126 targets Tsc1 and Cxcl12, mediate mTOR signaling or macrophage recruitment and activation, respectively.Suppression of either one in miR-126 deficient condition resulted in reduced bacteria burden.Although the importance of mTOR and CCR2 signaling in Mm infection in zebrafish models has been previously reported, the direct connection of these two pathways to miR-126 and its possible target tsc1 and cxcl12 is novel.However, the current study's significant weaknesses still need to be addressed.
Major concerns: 4, Figure 2 is problematic.tsc1a is the only one that showed significant upregulation upon miR-126 suppression.The remaining genes did not show significant changes upon Mm infection and miR-126 deletion.I suggest removing the non-significant data.LSA now encourages authors to provide a 30-60 second video where the study is briefly explained.We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608).Corresponding or first-authors are welcome to submit the video.Please submit only one video per manuscript.The video can be emailed to contact@life-science-alliance.org To upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plexYou will be guided to complete the submission of your revised manuscript and to fill in all necessary information.Please get in touch in case you do not know or remember your login name.
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You can contact the journal office with any questions, contact@life-science-alliance.orgAgain, congratulations on a very nice paper.I hope you found the review process to be constructive and are pleased with how the manuscript was handled editorially.We look forward to future exciting submissions from your lab.Sincerely, Eric Sawey, PhD Executive Editor Life Science Alliance http://www.lsajournal.org • Figure legends o Figure 1: Please correct the whole legend, miR-206?o Please clarify: "Bacterial burden analysis data points represent individual embryos (n=40-50 embryos per group) and are representative of 2 biological replicates".If each data point represents an individual embryo, then 40-50 biological replicates are depicted.Is this a representative plot for 2 experiments?
Please correct the whole legend, miR-206?Legend has been corrected o Please clarify: "Bacterial burden analysis data points represent individual embryos (n=40-50 embryos per group) and are representative of 2 biological replicates".If each data point represents an individual embryo, then 40-50 biological replicates are depicted.Is this a representative plot for 2 experiments?
Figure legends have been updated to clarify this point.
We have added new text to the discussion "We did not examine other markers of macrophage polarisation such as arg2 and nos2a which would allow further characterisation of the tnfa:gfp negative macrophage population in the miR-126 knockdown embryos" and added relevant references to Irg1 (Sanderson LE et al, 2015) Arg2 (Hammond FR et al, 2023) and Nos2a (Elks PM et al, 2013) as markers of leukocyte polarisation in zebrafish.

Figure 1 BFigure 1 E
Figure 1 B The legend is missing.We have doubled checked the manuscript document and it seems to be there: "Figure 1. Infection-induced miR-126 expression alters bacterial burden.(A) Expression of miR-126 following M. marinum infection analysed by qPCR at 1 and 3 dpi relative to uninfected embryos.(B) Expression of miR-126 in uninfected and infected, antagomir-injected (miR-126 knockdown) and scramble-injected embryos.(C) Representative images of M. marinum infection at 3 dpi in scramble control and miR-126 knockdown embryos.Scale bar represents 200 μm." Please take a moment to check your funder requirements.****Reviews, decision letters, and point-by-point responses associated with peer-review at Life Science Alliance will be published online, alongside the manuscript.If you do want to opt out of having the reviewer reports and your point-by-point responses displayed, please let us know immediately.**Thank you for your attention to these final processing requirements.Please revise and format the manuscript and upload materials within 7 days.Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.http://www.lsajournal.org---------------------------------------------------------------------------submittingyour Research Article entitled "Zebrafish tsc1 and cxcl12a increase susceptibility to mycobacterial infection".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.
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